Concerns about the safety of hormone therapy (HT) have prompted many women to use botanical dietary supplements as alternatives for the management of vasomotor symptoms related to menopause. As a result, many botanicals such as black cohosh and licorice that are being used by menopausal women rank within the top 40 in sales among all botanical dietary supplements in the United States. The safety of these popular botanical dietary supplements will be the focus of this project. We hypothesize that most botanical dietary supplements used by women to manage menopausal symptoms are safe, but that some botanicals might pose risks through interactions with therapeutic agents or through the formation of electrophilic metabolites. To test this hypothesis, the top botanical dietary supplements being used by menopausal women will be investigated for metabolic activation to potentially toxic metabolites, and the potential of these botanicals to cause drug-botanical interactions will be studied in vitro and then in a clinical study. Our three Specific Aims will include the following: 1) The metabolism and bioavailability of constituents of botanical dietary supplements popular among menopausal women will be tested using human liver microsomes, human intestinal microsomes, Caco-2 cells, and human hepatocytes. The structures of phase I and phase II metabolites of these botanical compounds will be determined, and the enzymes responsible for their formation will be identified. Possible formation of reactive metabolites of botanical constituents will be investigated in vitro using our innovative ultrafiltration UHPLC-MS/MS assay. The metabolism of botanical compounds in vitro will be confirmed in vivo using rats in collaboration with Project 2 and Core C. 2) The potential for drug-botanical interactions will be investigated in vitro according to FDA guidelines. Studies will include inhibition and induction of specific phase I and phase II enzymes and drug transporters that might cause adverse interactions with therapeutic agents. The compounds responsible for inhibition or induction of drug metabolizing enzymes and drug transporters will be identified in collaboration with Project 1, Core B and Core D. 3) Clinical safety studies of drug-botanical interactions will be carried out to test the hypothesis that botanical dietary supplements used by menopausal women might induce or inhibit cytochrome P450 enzymes involved in the phase I metabolism of therapeutic agents. Standardized extracts of hops, red clover and licorice that have been predicted to cause drug- botanical interactions during our preliminary in vitro studies will be administered to women in an effort to determine if they affect the metabolism of probe substrates of specific cytochrome P450 enzymes. Serum levels of botanical compounds and their metabolites will be measured to confirm the preclinical metabolism and absorption studies. Altogether, these in vitro and in vivo studies will produce preclinical and clinical data that will advance the understanding of the safety of botanical dietary supplements with respect to metabolism, bioavailability and the potential for botanical-drug interactions.